Dysregulation of programmed cell death is central to a number of human diseases. Insufficient cell death leads to cancer and some autoimmune diseases while excessive cell death contributes to a number of neurological disorders and AIDS. Overexpression of Bcl-2, an inhibitor of apoptosis, results from the chromosomal translocations characteristic of follicular lymphoma patients. Overexpression of a Bcl-2 family member, Bcl-XL, is frequently found in the Kaposi's sarcoma lesions of AIDS patients. Upregulation of one or more Bcl-2 family members in the late stage of tumor development is a common occurrence. However, the molecular mechanisms by which Bcl-2 family members regulate programmed cell death are only beginning to be understood. Again by unknown mechanisms Bcl-XL and Bcl-2 prevent the activation of caspases, a family of cysteine proteases that are key facilitators of apoptotic cell death. Recently, Bcl-2 family members were found to serve as caspase substrates. Caspases cleave Bcl-XL and Bcl-2 in the loop domain near the N-terminus which results in loss of the BH4 homology domain, a domain that is required for inhibition of cell death. As a result, cleavage of Bcl-XL and Bcl-2 by caspases converts these proteins from potent inhibitors of cell death to potent inducers of cell death. However, except for the universally conserved aspartate at the P1 position of the Bcl-XL cleavage sites, these sites do not resemble any other known caspase cleavage sites. Experiments are proposed to determine if posttranslational modification of the Bcl-XL cleavage sites by cellular kinases/phosphatases modulates the recognition of these site by caspases. The role of phosphorylation and the responsible kinases that regulate BcI-XL function during apoptosis will be studied in neurons and tumor cell lines. The protein domains required for the pro-death activity of cleaved Bcl-XL will be determined by extensive mutagenesis. Biochemical and functional analyses will be performed to explore the molecular mechanism behind this pro-apoptotic activity. These studies are expected to significantly advance our understanding of the molecular processes of programmed cell death which impact on a wide range of human disorders.